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Supplementary Material 1

There are many reports for andrographolide modification regarding antitumor effects. Transformation of the five-membered lactone ring to furan aromatic ring still results in compounds with good cytotoxicity. To determine further the importance of the five-membered lactone ring and to obtain better lead compounds, we transformed the five-membered lactone ring in andrographolide. New types of ent-labdane diterpene derivatives were made, whose cytotoxic activities were measured in vitro. Preliminary SAR was summarized and two compounds, 7 and 26, with good cytotoxic activity were obtained, which have the potential to be developed into new antitumor drugs.

Jolkinolides, isolated from Euphorbia fischeriana Steud, are naturally occurring tetracyclic ent-abietane diterpenes, some of which exhibit promising antitumor and other biological activity. An efficient strategy for the synthesis of jolkinolides A and B is described starting from readily available steviol in 10 and 11 steps with total yields of over 10%, respectively.

Extracted from Euphorbia, ent-Abietane lactones can be classified into several categories, such as Jolkinolides and Helioscopinolides, according to their structural features. The study of ent- Abietane lactones could date back to 1972, when Jolkinolide A and B were first isolated from Euphorbia jolkini Boiss. Since then, many other ent-Abietane lactones have been extracted from different species of Euphorbia. Their bio-activities include anti-tumor activity, anti-inflammatory activity as well as anti-bacterial activity. Among them, derivatives of Jolkinolide B draw the most attention for their high anti-tumor activity. There are many studies focus on the syntheses of Jolkinolides. In 1989, the first and efficient synthesis of Jolkinolides was accomplished by Katsumura et al. Their strategy to construct the last ring of Jolkinolides contributes a lot to the following studies. In the following thirty years, there are also other semi-syntheses of Jolkinolides conducted, basing on different starting materials. In this review, we will give a brief clarification of ent-Abietane lactones, as well as their bio-activities and syntheses.

CPUK02 (15-Oxosteviol benzyl ester) is a new semi-synthetic of stevioside. The anticancer activity was evaluated in several in vitro and in vivo models, and the mechanism of bioactivity studied. The anti-proliferation activity in vitro was tested by the MTT assay, which showed that CPUK02 strongly inhibited cell proliferation against a variety of human cancer cell lines. The human liver cancer cell line was significantly more sensitive than the normal liver cell line when CPUK02 was administered at low concentration. In several mouse bearing human cancer xenograft models, CPUK02 demonstrated comparable anti-tumor effects to fluorouracil (5-FU), but showed lower toxic effects. Annexin/PI double staining flow cytometry and DAPI staining experiments were used to observe the cellular morphological changes caused by CPUK02, which established that the compound induces cell apoptosis, and that the apoptotic effects appeared a dose dependent relationship. Western blot experiments were also carried out to explore the mechanism of this compound. It was found that this compound can activate the apoptosis signal cascade via the mitochondrial pathway in cells. These results indicate that CPUK02 may be a promising new compound with notable in vitro and in vivo anticancer activity. Further studies to investigate the effects on treating tumors are under way.

Since two asymmetric centres are usually present in the bisbenzylisoquinolines, the circular dichroism is known to be an important method to elucidate their stereochemistry. We studied the relationships of the CD spectra with the stereochemistry of synthetic RR-(1), SS-(2), RS-daurisoline (3) and SR-daurisoline (4). By comparision of the CD spectrum of natural daurisoline (1) from Menispermum dauricum (Menispermaceae) with those of synthetic enantiomers, the absolute configuration was established as RR-daurisoline (1).

A series of novel 7′-azaindirubin ( 1a – g ) and 7-azaindirubin ( 2a , 2c , 2e and 2f ) derivatives were designed and synthesized. Their structures were characterized by 1 H NMR and MS spectroscopy as well as by elemental analysis. Their inhibitory properties against CDK2/cylinA were evaluated in vitro . In contrast to indirubin, some of the described azaindirubins emerged as potent inhibitors of CDK2/cylinA and compound 2b had more potent activity. Biological tests also showed that nitrogen atom at 7-position of azaindirubin was more beneficial to enhance the kinase inhibitory activity.

A series of 2-alkylbenzimidazole derivatives 9a – n have been designed and synthesized as a novel class of non-peptide angiotensin II AT 1 receptor antagonists. The synthesized compounds were evaluated for their antagonism of angiotensin II, induced contraction in the rabbit thoracic aortic ring and the results showed that compounds 9a , 9g and 9j exhibited potent antagonistic activity of AT 1 receptor.

The cytotoxic activities of (I) and (II) are evaluated showing that (II) has medium antiproliferative activity.

Twenty 7-azaindirubin derivatives were designed and synthesized. Their antitumor activities were evaluated in vitro against DU145 cell line. The pharmacological results showed that most of the prepared compounds displayed the excellent activity. Compound 18 exhibited the most potent antitumor activity among the tested compounds.

A series of novel derivatives of ligustrazine linked with substituted benzoyl guanidine were synthesized. These compounds have not been reported in literature, and their chemical structures were confirmed by IR, 1H NMR and MS. The results of NHE1 inhibitory activity test showed that compounds I2, I3, I4, I6, and I7 possess more potent NHE1 inhibitory activity than cariporide.

Aim To discover more active and water-soluble derivatives of tetracyclic diterpenoids containing an exo-methylene cyclopentanone or an α-methylenelactone moiety. Methods All of the key intermediates were synthesized from stevioside, and the target compounds were obtained through glycosylation of the 4-carboxyl group. The cytotoxicity of the target compounds against six human cancer cell lines, HepG2, Bel-7402, A549, U251, MCF-7 and MDA-MB-231, were evaluated by the MTT assay. Results Compound 1b was more effective than the positive control adriamycin against the HepG2, Bel-7402, A549, MCF-7, and MDA-MB-231 cell lines with IC50 values of 0.12, 0.91, 0.35, 0.08, and 0.07 μmol·L−1, respectively. Moreover, compound 3c exhibited the most potent and selective cytotoxic activity against the HepG2 cell line (IC50, 0.01 μmol·L−1). Conclusion Compounds 1b and 3c could be considered as potential anticancer candidates for further study.

A series of tetracyclic diterpenoids bearing the α-methylenelactone group have been synthesized and screened for their in vitro anti-tumor activities against six human cancer cell lines. The results showed that compounds 1c, 2a and 2b exhibited significant cytotoxicity superior to the positive control doxorubicin hydrochloride against MDA-MB-231, K562 and HepG2 cell lines. In particular, compound 2b was identified as the most promising anticancer agent against HepG2 cells with IC(50) value of 0.09μM.

To synthesize a series of 2-arylimino-4-thiazolidone derivatives and 2-imidazolino [2,3-b]-4-thiazolidone in order to get some novel potent compounds with nitric oxide synthases (NOS) inhibitory activity.The target compounds were prepared by reaction of N-chloroacetyl-1,2,3,4-tetrahydroisoquinoline or N-chloroacetylphthalimide with substituted thioureas, their NOS inhibitory activity were measured.The 15 new compounds were synthesized and most of the reaction yields were over 65%. The structures of new compounds were identified by IR, 1H NMR, MS and elemental analyses. Bioassay indicated that, most of 15 new compounds synthesized had confirmed bioactivities inhibition against NOS.

A series of 2-alkylbenzimidazoles bearing a N -phenylpyrrole moiety were synthesized and evaluated as a novel class of AT 1 receptor antagonists. Among them, compounds 10a and 10g inhibited [ 125 I] AngII-binding affinity to AT 1 receptor at nanomolar level and potently inhibited the Ang II-induced pressor response by oral administration. Moreover, evaluation in spontaneously hypertensive rats showed that 10a is an orally active AT 1 receptor antagonist.

Based on ticlopidine active as an ADP receptor antagonist for inhibiting platelet aggregation in clinical test, and upon finding (±)-1,2-substituted-7-sulfonylamide/amide-1,2,3,4-tetrahydroisoquinoline ( 11 – 31 ) inhibited of platelet aggregation, a series of (±)-1- o -chlorophenyl-2-substituted-tetrahydroisoquinoline derivatives was designed and synthesized. Four analogs proved to be potential antiplatelet aggregation agents, and compound 9 (TQP-3, applying for patent) which inhibits ADP-induced human platelet aggregation with IC 50 values of approximately 0.206 nM was the most active. Compound 2 is more active than compound 1 , which (Type I) is similar to ticlopidine. This is because there is a spacial hindrance in compound 1 , and the o -chloro group of compound 2 may play the same a role as o -chloro group of ticlopidine. On the other hand, with the different substitutions at different positions on the 2-substituted phenylacyl group, their inhibition of platelet aggregation differs. These compounds with m -substituted group ( 5 , 7 , 9 ) showed a higher IC 50 value for inhibiting ADP-induced human platelet aggregation than those with o -substituted group ( 4 , 6 ) or p -substituted group ( 3 , 8 ). It was observed that their inhibition is bromine-substituted derivative ( 9 ), chlorine-substituted derivative ( 7 ), and nitro-substituted derivative ( 5 ) in turn. Moreover, these compounds (Type II) may be more similar to clopidogrel than to ticlopidine due to the acyl group at 2 position of the nucleus playing a role as the ester group of clopidogrel. It was conjectured that these analogs function as a potential antiplatelet aggregation role by acting as ADP receptor antagonists.

To search for novel compounds with potent nNOS inhibitory activity for the treatment of Alzheimer's disease.The target compounds were obtained by introducing benzenealkyl groups into the structure of isothioureas. nNOS inhibitory activity assays were conducted for the target compounds.Sixteen benzenealkyl isothiourea compounds (I1-16) were synthesized by three different synthetic methods from benzylamine (1) or (substituted) phenethylamine (2). Compounds I1-6 were synthesized from 1 or 2 by reaction with benzoyl isothiocyanate to form the corresponding benzoylthioureas 3 or 4, followed by hydrolysis with 10% sodium hydroxide solution, then S-alkylation with methyl iodide or ethyl iodide. I7-14 were synthesized from 1 or 2 by reaction with methyl isothiocyanate to form the corresponding 1, 3-disubstituted thioureas 7 or 8 which were S-alkylated with methyl iodide or ethyl iodide. I15 and I16 were synthesized from 2 by reaction with dimethyl cyanodithioimidocarbonate. The structures of compounds I1-16 were confirmed by MS, IR, 1HNMR and elementary analysis. The results of preliminary pharmacological test showed that all compounds possessed nNOS inhibitory activity, among which compounds I8, I12 and I14 had good activity.Compounds I8, I12 and I14 showed superior pharmacological profiles to the control compound S-methyl-N-(4-methoxyphenyl) isothiourea. The IC50 values of compounds I8, I12 and I14 inhibiting nNOS were 8.13 x 10(-7) mol.L-1, 1.74 x 10(-7) and 2.23 x 10(-7) mol.L-1 respectively, and it is worth further studying.

It has been shown that independent sources of nitric oxide (NO) and the inflammatory cytokine tumor necrosis factor alpha (TNFα) contribute to the breakdown of the blood-brain barrier (BBB) in the pathogenesis of a number of brain disorders. However, the interaction of NO and TNFα has not been elucidated. The present study was designed to determine whether the toxicity induced by NO is altered by TNFα in brain capillary endothelial cells (BCECs), and if so, whether it is related to the generation of Superoxide. TNFα (50–400 U ml ) did not produce toxicity until at a concentration of 800 U ml . This toxic effect was completely blocked by copper-zinc Superoxide dismutase (SOD)/catalase or Nω-nitro-L-arginine methyl ester (L-NAME) or oxyhemoglobin (HbO2). Sodium nitroprusside (SNP) reduced with 0.4 mM ascorbate (SNP/Vc) significantly increased Lactate dehydrogenase (LDH) efflux in a concentration-dependent manner. This cytotoxicity of SNP/Vc was also completely inhibited by SOD/catalase or HbO2. When SNP/Vc used in combination with 400 U ml TNFα, a more remarkable LDH efflux was induced than SNP/Vc alone, even as little as 0.01mM SNP/Vc was toxic, although a dose of 400 U ml TNFα alone had no effect on LDH efflux. In addition, either 0.4 mM SNP/Vc and 800 U ml TNFa alone or 0.4 mM SNP/Vc and 400 U ml TNFα in combination significantly increased malondialdehyde (MDA) content, but nitric oxide synthase (NOS) activity was inhibited only by SNP/Vc and TNFa in combination. These results suggest that TNFα enhances the toxicity of NO in BCECs and that at least part of this enhancement involves the generation of Superoxide.